Bifluorescence detection method for detecting gene expression in filamentous fungi and exogenous gene expression method

Abstract

The invention discloses a bifluorescence detection method for detecting gene expression in filamentous fungi and an exogenous gene expression method. The invention provides a double-fluorescence detection system and a double-fluorescence detection method capable of efficiently and rapidly detecting the expression efficiency of different promoters and insertion site genes of filamentous fungi, the influence caused by the growth difference of strains can be eliminated, the quantitative detection of the gene expression condition is realized, and the advantages of high efficiency and accuracy are achieved. Six efficient insertion sites are screened out, more possibilities are provided for successful heterologous expression of complex high-value natural products in aspergillus nidulans, and high application value is achieved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a dual fluorescence detection method for detecting gene expression in filamentous fungi and an exogenous gene expression method. Background Art

[0002] Aspergillus nidulans, a model organism for fungal research, was one of the first to be sequenced. Its genetic background is well-established, and genetic manipulation techniques are highly mature, making it a common target for heterologous expression studies of natural products. Aspergillus nidulans, as a heterologous expression host, can accurately identify and remove intron sequences from fungal genes. It is also well-suited for expressing complex eukaryotic proteins, particularly those difficult to express in Escherichia coli. It offers advantages such as strong growth, ease of cultivation, and low cost.

[0003] For example, the invention application with publication number CN108588060A discloses a recombinant oxalate decarboxylase expressed in a filamentous fungal host cell, wherein the recombinant filamentous fungal host cell comprises one or more copies of an oxalate decarboxylase expression cassette integrated into its genome, the oxalate decarboxylase expression cassette comprising a promoter, a signal peptide coding sequence, an oxalate decarboxylase encoding gene, and a terminator. The host cell can be constructed by either random integration or site-specific integration. The filamentous fungus is a strain of Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, or Aspergillus awamori.

[0004] When using Aspergillus nidulans as a heterologous expression host, commonly used promoters to initiate exogenous gene expression include the constitutive promoter gpdAp, the inducible promoter starch-inducible promoter amyBp, the xylose-inducible promoter xlyp, etc., but there has been no specific comparison of the efficiency of these promoters.

[0005] In current research, there are relatively few sites available for inserting foreign genes in Aspergillus nidulans as a heterologous expression host. The yA site and the wA site are commonly used. When foreign genes are inserted into these two sites, the color of the Aspergillus nidulans spores changes from green to yellow and white, respectively. The change in spore color can increase the accuracy of transformant screening ([1] Mayorga, ME; Timberlake, WE The developmentally regulated Aspergillus nidulan swa gene encodes a polypeptide homologous to polyketide and fatty acid synththases. Mol. Gen. Genet. 1992, 235, 205-212. [2] Clutterbuck, AJ Absence of laccase from yellow-spored mutants of Aspergillus nidulans. J. Gen. Microbiol. 1972. 70. 423-435.).

[0006] However, when heterologously expressing complex natural product biosynthetic gene clusters, multiple genes are often present, requiring multiple sites to accommodate the insertion of these different genes. This lack of available sites has limited research on heterologous expression of natural products in Aspergillus nidulans. Therefore, identifying other sites in Aspergillus nidulans suitable for exogenous gene insertion has important application value for the heterologous expression of some high-value natural products. Summary of the Invention

[0007] In view of the above-mentioned deficiencies in the prior art, the present invention provides a dual fluorescence detection method for detecting gene expression in filamentous fungi and an exogenous gene expression method.

[0008] The present invention first provides a dual fluorescence detection method for detecting gene expression in filamentous fungi, comprising the following steps:

[0009] (1) inserting a gene for expressing a first fluorescent protein into a first insertion site of the filamentous fungus, inserting a gene for expressing a second fluorescent protein into a second insertion site of the filamentous fungus, or inserting an exogenous expression plasmid and transferring the exogenous expression plasmid into the filamentous fungus to obtain a recombinant filamentous fungus;

[0010] (2) culturing the recombinant filamentous fungus obtained in step (1), detecting the fluorescence intensity of the first fluorescent protein and the second fluorescent protein, and using the ratio of the fluorescence intensity of the second fluorescent protein to the fluorescence intensity of the second fluorescent protein as the expression efficiency of the second fluorescent protein,

[0011] Wherein, the fluorescence colors of the first fluorescent protein and the second fluorescent protein are different;

[0012] If the effect of the insertion site on gene expression needs to be detected, the insertion site to be detected is used as the second insertion site;

[0013] If the effect of a promoter on gene expression is to be detected, the promoter to be detected is used as the promoter of a gene for expressing a second fluorescent protein and is inserted into the second insertion site of the filamentous fungus;

[0014] If what needs to be detected is the effect of the expression plasmid on gene expression, the expression plasmid to be detected is used as an exogenous expression plasmid for expressing the gene of the second fluorescent protein.

[0015] Preferably, the filamentous fungus is Aspergillus, Penicillium or Trichoderma.

[0016] More preferably, the filamentous fungus is Aspergillus niger (A. niger), Aspergillus nidulans (A. nidulans), Aspergillus oryzae (A. oryzae), Aspergillus fumigatus (A. fumigatus) or Aspergillus awamori (A. awamori);

[0017] or the filamentous fungus is Penicillium citrinum of the genus Penicillium;

[0018] Or the filamentous fungus is Trichoderma reesei of the genus Trichoderma.

[0019] Most preferably, the filamentous fungus is Aspergillus nidulans (A. nidulans) of the genus Aspergillus. The Aspergillus nidulans strain is preferably Aspergillus nidulans LO8030 (Chiang YM, Ahuja M, Oakley CE, et al. Development of Genetic Dereplication Strains in Aspergillus nidulans Results in the Discovery of Aspercryptin [J]. Angewandte Chemie International Edition, 2015, 55 (5): 1694-1697.). The first insertion site is the yA site or the wA site, and the second insertion site is any one of the following sites:

[0020] (1) Site 2: The upstream and downstream genes in Aspergillus nidulans are AN0716 and AN9436, and the chromosome position of the site in Aspergillus nidulans is ChrVIII: 2657115-2689534;

[0021] (2) Site 6: The upstream and downstream genes in Aspergillus nidulans are AN4560 and AN4777, and the chromosomal location of the site in Aspergillus nidulans is ChrIII: 1022424-1662379;

[0022] (3) Locus 20: The upstream and downstream genes in Aspergillus nidulans are AN0104 and AN0067, and the chromosome position of the locus in Aspergillus nidulans is ChrVIII: 4602068-4707084;

[0023] (4) AU1 locus: The upstream and downstream genes in Aspergillus nidulans are AN8385 and AN8378, and the chromosome position of the locus in Aspergillus nidulans is ChrIII: 293891-319970;

[0024] (5) F9 locus: The upstream and downstream genes in Aspergillus nidulans are AN7916 and AN7905, and the chromosome position of the locus in Aspergillus nidulans is ChrIII: 198368-226926;

[0025] (6) MON locus: The upstream and downstream genes in Aspergillus nidulans are AN0153 and AN10039, and the chromosomal position of the locus in Aspergillus nidulans is ChrIII: 4435547-4460859.

[0026] Preferably, the first fluorescent protein and the second fluorescent protein are each selected from the following: red fluorescent protein, green fluorescent protein, blue fluorescent protein, and yellow fluorescent protein. In the present invention, the two fluorescent proteins only need to be able to distinguish the first fluorescent protein from the second fluorescent protein. Of course, for more convenient detection, the two fluorescent proteins may have a high degree of differentiation so that they can be easily distinguished and detected. For example, red and green dual fluorescence can be used.

[0027] Preferably, when inserting the gene for expressing the first fluorescent protein into the first insertion site of the filamentous fungus and inserting the gene for expressing the second fluorescent protein into the second insertion site of the filamentous fungus, homologous recombination is used for insertion.

[0028] The present invention further provides a method for expressing an exogenous gene, using a filamentous fungus as a heterologous expression host, and inserting the exogenous gene to be expressed into any one of the following sites:

[0029] (1) Site 2: The upstream and downstream genes in Aspergillus nidulans are AN0716 and AN9436, and the chromosome position of the site in Aspergillus nidulans is ChrVIII: 2657115-2689534;

[0030] (2) Site 6: The upstream and downstream genes in Aspergillus nidulans are AN4560 and AN4777, and the chromosomal location of the site in Aspergillus nidulans is ChrIII: 1022424-1662379;

[0031] (3) Locus 20: The upstream and downstream genes in Aspergillus nidulans are AN0104 and AN0067, and the chromosome position of the locus in Aspergillus nidulans is ChrVIII: 4602068-4707084;

[0032] (4) AU1 locus: The upstream and downstream genes in Aspergillus nidulans are AN8385 and AN8378, and the chromosome position of the locus in Aspergillus nidulans is ChrIII: 293891-319970;

[0033] (5) F9 locus: The upstream and downstream genes in Aspergillus nidulans are AN7916 and AN7905, and the chromosome position of the locus in Aspergillus nidulans is ChrIII: 198368-226926;

[0034] (6) MON locus: The upstream and downstream genes in Aspergillus nidulans are AN0153 and AN10039, and the chromosomal position of the locus in Aspergillus nidulans is ChrIII: 4435547-4460859.

[0035] Preferably, the filamentous fungus is Aspergillus, Penicillium or Trichoderma.

[0036] More preferably, the filamentous fungus is Aspergillus niger (A. niger), Aspergillus nidulans (A. nidulans), Aspergillus oryzae (A. oryzae), Aspergillus fumigatus (A. fumigatus) or Aspergillus awamori (A. awamori);

[0037] or the filamentous fungus is Penicillium citrinum of the genus Penicillium;

[0038] Or the filamentous fungus is Trichoderma reesei of the genus Trichoderma.

[0039] More preferably, the filamentous fungus is Aspergillus nidulans (A. nidulans) of the genus Aspergillus. The Aspergillus nidulans species used is preferably Aspergillus nidulans LO8030.

[0040] In this application, the primary insertion site is the site where the gene is determined to be inserted and serves as an internal reference site. For example, in Aspergillus nidulans, the yA or wA sites can be selected because disruption of these sites changes spore color, improving transformant screening efficiency. The secondary insertion site is the site to be tested for gene insertion.

[0041] The present invention measures the expression efficiency of 17 sites in Aspergillus nidulans and compares the expression efficiencies of these sites. When Aspergillus nidulans is selected as a heterologous expression host, more insertion sites are provided for the efficient expression of foreign genes.

[0042] The present invention provides a dual-fluorescence detection system and method for rapidly and efficiently detecting gene expression efficiencies from different promoters and insertion sites in filamentous fungi. This eliminates the influence of strain growth differences, enabling quantitative detection of gene expression with high efficiency and accuracy. The system screened for six highly efficient insertion sites, expanding the possibilities for successful heterologous expression of complex, high-value natural products in Aspergillus nidulans and demonstrating high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the map of the red fluorescent protein plasmid pZR-1 at the yA site.

[0044] Figure 2 This is the map of the yA site green fluorescent protein plasmid pZR-2.

[0045] Figure 3 This is a map of the insertion site of the green fluorescent protein plasmid pZR-X.

[0046] Figure 4 Schematic diagram of electrophoresis results for interface verification at different sites.

[0047] Figure 5 Schematic diagram of the red and green dual fluorescence detection method.

[0048] Figure 6 Schematic diagram of the gene expression efficiency detection results at different insertion sites. DETAILED DESCRIPTION

[0049] The present invention aims to construct a red-green dual fluorescence detection system for efficiently and accurately measuring gene expression efficiency in filamentous fungi, which can be used to detect and evaluate the gene expression efficiency of different gene insertion sites, free plasmids, and different promoters.

[0050] First, the present invention uses Aspergillus nidulans LO8030 as the starting strain. By comparing with essential genes in Pichia pastoris, homologous genes were found in Aspergillus nidulans LO8030, and 20 different insertion sites were identified. In addition, the location of the biosynthetic gene cluster of 8 major secondary metabolites knocked out in Aspergillus nidulans (Chiang YM, Ahuja M, Oakley CE, et al. Development of Genetic Dereplication Strains in Aspergillus nidulans Results in the Discovery of Aspercryptin [J]. Angewandte Chemie International Edition, 2015, 55 (5): 1694-1697.) was also identified as a site that needs to be screened. Because the production of these compounds themselves is high, it is believed that the expression efficiency of the genes located at this location is also relatively strong. Therefore, a total of 28 sites that need to be tested were identified.

[0051] Secondly, the present invention uses red and green dual fluorescent proteins to detect the gene expression efficiency of these different sites. Select the yA site as the control site, first insert the red fluorescent protein mcherry at the yA site, then insert a green fluorescent protein eGFP at the site to be tested and the yA site respectively, measure the green fluorescence intensity and the red fluorescent protein intensity in different mutants by microplate reader, and utilize their ratio to reflect the gene expression efficiency in each site. Due to the presence of the red fluorescent protein internal reference gene, the impact of the different growth conditions of the thalline on the experimental results can be avoided, and the purpose of high efficiency and accuracy is achieved. The insertion of different fluorescent protein genes has selected the method of homologous recombination, and by constructing a plasmid, a gene cassette is obtained. The yA site red fluorescent protein insertion plasmid with the upstream homology arm and the downstream homology arm of the yA site and with a promoter, a red fluorescent protein gene, a terminator, and an auxotrophic marker is constructed. The required gene cassette is obtained by the mode of PCR amplification, and is imported into the yA site by the mode of protoplast transformation. Similarly, a green fluorescent protein insertion plasmid was constructed with upstream and downstream homology arms at different insertion sites, along with a promoter, green fluorescent protein gene, terminator, and auxotrophic marker. After obtaining the gene cassette, protoplast transformation experiments were performed, successfully generating mutants expressing red fluorescent protein and green fluorescent protein at 17 different sites and one yA site.

[0052] Finally, the green fluorescence intensity was measured using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The red fluorescence intensity was measured at an excitation wavelength of 570 nm and an emission wavelength of 610 nm. The green fluorescent protein and red fluorescent protein intensities of 20 different strains were measured in LMM medium at 37°C and 180 rpm for 0, 6, 12, 18, 24, and 36 hours. By comparing their ratios with the ratio of the yA site, six highly efficient insertion sites were screened, providing more possibilities for the successful heterologous expression of complex, high-value natural products in Aspergillus nidulans and demonstrating high application value.

[0053] Example 1: Screening of foreign gene insertion sites in Aspergillus nidulans

[0054] According to the amino acid sequence of the essential gene in Pichia pastoris provided in the literature (Gao J, Zuo Y, Xiao F, et al. Biosynthesis of catharanthine in engineered Pichia pastoris [J]. Nature Synthesis, 2023, 2: 231-242. DOI: 10.1038 / s44160-022-00205-2.), a BLAST comparison was performed with the amino acid sequence of Aspergillus nidulans LO8030 to find the homologous gene in Aspergillus nidulans (see Table 1).

[0055] Based on the different chromosomal locations of these genes and the requirement that the distance between two essential genes be greater than 600 base pairs (to ensure that the essential genes on either side are not damaged during genetic manipulation), the researchers identified sites between two adjacent essential genes as highly suitable for exogenous gene insertion. Bioinformatics analysis identified 20 sites that met these criteria, designated sites 1-20.

[0056] Then, the locations of the gene clusters of the eight major secondary metabolites knocked out in Aspergillus nidulans LO8030 were searched, and their locations were determined. They were named as ST, AU1, TE, EM, ASP, AU2, F9, and MON sites according to the different secondary metabolites knocked out.

[0057] The detailed information of the 28 exogenous gene insertion sites screened in Aspergillus nidulans is shown in Table 1.

[0058] Table 1 28 foreign gene insertion sites screened in Aspergillus nidulans

[0059]

[0060]

[0061] a Essential genes of Pichia pastoris studied in the literature (Gao J, Zuo Y, Xiao F, et al. Biosynthesis of catharanthine inengineered Pichia pastoris [J]. Nature Synthesis, 2023, 2: 231-242. DOI: 10.1038 / s44160-022-00205-2.).

[0062] b Homologous genes of Pichia pastoris genes or eight Aspergillus nidulans secondary metabolite gene cluster genes.

[0063] Example 2: Construction of an Aspergillus nidulans strain expressing dual fluorescent proteins

[0064] After determining the sites to be tested, the present invention has created a new system for detecting the gene expression efficiency of these sites. In order to obtain gene cassettes with different insertion fragments, yeast homologous recombination technology was used to connect multiple fragments to construct plasmids. First, a plasmid pZR-1 with a red fluorescent protein yA site insertion fragment was constructed (see the plasmid map). Figure 1 The plasmid backbone genes fragment includes a 2μ sequence, URA3, an AMP resistance screening gene, and ori, gpdAp is a promoter, yA-up is an upstream homology arm of the yA site, yA-down is a downstream homology arm of the yA site, mCherry is a red fluorescent protein gene, CYC1terminator is a terminator, Apara pyrG is a uracil-uridine auxotrophic marker gene, AfriboB is a riboflavin auxotrophic marker gene, and EGFP is a green fluorescent protein gene. The target fragment is then amplified by PCR (primers used for PCR amplification: upstream primer yA-F: 5'-gatggagattggcgccgttc-3' and downstream primer yA-R: 5'-atcggcgttgagcgcgtcat-3'). After rubber tapping to recover 8-10 μg of the gene cassette fragment, the red fluorescent protein is inserted into the yA site of Aspergillus nidulans using PEG-mediated protoplast transformation. The transformant whose spore color changed from green to yellow was verified at the molecular level and named LO8030-yA-cherry after confirmation of its correctness.

[0065] Then, based on LO8030-yA-cherry, the green fluorescent protein eGFP was also transferred into the yA site. Similarly, a yA site insertion fragment plasmid pZR-2 with green fluorescent protein was first constructed (see the plasmid map for details). Figure 2 The plasmid backbone genes fragment includes gpdAp as a promoter, yA-down as a homology arm downstream of the yA site, CYCl terminator as a terminator, Apara pyrG (the total length is 1997 bp, and the plasmid selects the 858 bp-1997 bp part as the homology arm) as a uracil-uridine auxotrophic marker gene, AfriboB as a riboflavin auxotrophic marker gene, and EGFP as a green fluorescent protein gene. The primers used for PCR amplification are Apara pyrG (the upstream primer is Apara pyrG), AfriboB (the upstream primer is Apara pyrG), and EGFP (the upstream primer is Apara pyrG). After the target fragment size and sufficient concentration of the gene cassette were obtained by rubber tapping and recovery (pyrG-F: 5'-ctcacatcgatatcctctcc-3' and downstream primer yA-R: 5'-atcggcgttgagcgcgtcat-3'), protoplast transformation was performed, and the obtained transformed strain was verified at the molecular level interface. After confirmation of its correctness, it was named LO8030-yA-cherry-eGFP. Thus, the control strain of the yA site was successfully obtained.

[0066] According to the 28 different sites screened out, the gene expression efficiency of these sites is measured, and correct results can be obtained quickly and accurately by the red-green dual fluorescence detection method of the present invention. On the basis of LO8030-yA-cherry, green fluorescent protein is inserted into the different sites that need to be detected, and then the difference in expression efficiency between these different sites can be quickly obtained according to the ratio of the intensity of green fluorescent protein and red fluorescent protein intensity. The reason why red fluorescent protein is inserted into the yA site is to compare it as an internal reference, so that the impact of the difference in the spontaneous growth of Aspergillus nidulans fungi on the accuracy of the experimental results can be avoided, and convenience is also provided for the detection of subsequent fluorescence intensity, arriving at the purpose quickly.

[0067] 28 corresponding plasmids with upstream and downstream homology regions and eGFP green fluorescent protein were constructed for the insertion fragments of these 28 sites, and were named pZR-3, pZR-4, ..., pZR-30 (Table 1). The plasmid maps are shown in Figure 3 ,in Figure 3 In pZR-X, X-up, and X-down, X represents the site names 3, 4, ..., 30. Gene cassettes were obtained by PCR for protoplast transformation. After interface verification of the obtained different transformants, 17 transformants with different sites, including 2, 3, 4, 6, 8, 13, 16, 18, 20, ST, AU1, TE, EM, ASP, AU2, F9, and MON, were successfully obtained. The interface verification results are shown in the figure below. Figure 4 shown.

[0068] Example 3: Fluorescence intensity detection of transformed strains with different insertion sites

[0069] The fluorescent protein intensity of the obtained different strains was detected by measuring green fluorescent protein and red fluorescent protein at different wavelengths using an enzyme marker. Figure 5 Schematic diagram of the red and green dual fluorescence detection method.

[0070] First, add 100 μL of LMM medium to a black 96-well plate, then add 100 μL of spore suspension of the corresponding strains at different sites, and culture at 37 ° C and 180 rpm. The green fluorescent protein intensity and red fluorescent protein intensity of these strains were measured at 0, 6, 12, 18, 24, and 36 hours respectively. The green fluorescent protein was detected at an excitation wavelength of 485 nm and an emission wavelength of 528 nm, and the red fluorescent protein was detected at an excitation wavelength of 570 nm and an emission wavelength of 610 nm. There were 3 transformed strains at each site, and each experiment was repeated 3 times. By the formula: E (位点表达效率) = green fluorescence intensity / red fluorescence intensity to detect the difference in gene expression intensity between the 17 sites to be tested and the yA site. The results are as follows Figure 6 As shown, the gene expression efficiency of 6 sites was higher than that of yA site, namely sites 2, 6, 20, MON, AU1, and F9.

Claims

1. A dual fluorescence detection method for detecting gene expression in filamentous fungi, characterized in that: The following steps are involved: (1) inserting a gene for expressing a first fluorescent protein into a first insertion site of the filamentous fungus, inserting a gene for expressing a second fluorescent protein into a second insertion site of the filamentous fungus, or inserting an exogenous expression plasmid and transferring the exogenous expression plasmid into the filamentous fungus to obtain a recombinant filamentous fungus; (2) culturing the recombinant filamentous fungus obtained in step (1), detecting the fluorescence intensity of the first fluorescent protein and the second fluorescent protein, and using the ratio of the fluorescence intensity of the second fluorescent protein to the fluorescence intensity of the second fluorescent protein as the expression efficiency of the second fluorescent protein, Wherein, the fluorescence colors of the first fluorescent protein and the second fluorescent protein are different; If the effect of the insertion site on gene expression needs to be detected, the insertion site to be detected is used as the second insertion site; If the effect of a promoter on gene expression is to be detected, the promoter to be detected is used as the promoter of a gene for expressing a second fluorescent protein and is inserted into the second insertion site of the filamentous fungus; If what needs to be detected is the effect of the expression plasmid on gene expression, the expression plasmid to be detected is used as an exogenous expression plasmid for expressing the gene of the second fluorescent protein.

2. The dual fluorescence detection method for detecting gene expression in filamentous fungi according to claim 1, characterized in that: The filamentous fungus is Aspergillus, Penicillium or Trichoderma.

3. The dual fluorescence detection method for detecting gene expression in filamentous fungi according to claim 2, characterized in that: The filamentous fungus is Aspergillus niger (A. niger), Aspergillus nidulans (A. nidulans), Aspergillus oryzae (A. oryzae), Aspergillus fumigatus (A. fumigatus) or Aspergillus awamori (A. awamori) of the genus Aspergillus; or the filamentous fungus is Penicillium citrinum of the genus Penicillium; Or the filamentous fungus is Trichoderma reesei of the genus Trichoderma.

4. The dual fluorescence detection method for detecting gene expression in filamentous fungi according to claim 3, characterized in that: The filamentous fungus is Aspergillus nidulans (A. nidulans) of the genus Aspergillus.

5. The dual fluorescence detection method for detecting gene expression in filamentous fungi according to claim 4, characterized in that: The first insertion site is the yA site or the wA site, The second insertion site is any one of the following sites: (1) Site 2: The upstream and downstream genes in Aspergillus nidulans are AN0716 and AN9436, and the chromosome position of the site in Aspergillus nidulans is ChrVIII: 2657115-2689534; (2) Site 6: The upstream and downstream genes in Aspergillus nidulans are AN4560 and AN4777, and the chromosomal location of the site in Aspergillus nidulans is ChrIII: 1022424-1662379; (3) Locus 20: The upstream and downstream genes in Aspergillus nidulans are AN0104 and AN0067, and the chromosome position of the locus in Aspergillus nidulans is ChrVIII: 4602068-4707084; (4) AU1 locus: The upstream and downstream genes in Aspergillus nidulans are AN8385 and AN8378, and the chromosome position of the locus in Aspergillus nidulans is ChrIII: 293891-319970; (5) F9 locus: The upstream and downstream genes in Aspergillus nidulans are AN7916 and AN7905, and the chromosome position of the locus in Aspergillus nidulans is ChrIII: 198368-226926; (6) MON locus: The upstream and downstream genes in Aspergillus nidulans are AN0153 and AN10039, and the chromosomal position of the locus in Aspergillus nidulans is ChrIII: 4435547-4460859.

6. The dual fluorescence detection method for detecting gene expression in filamentous fungi according to claim 1, characterized in that: The first fluorescent protein and the second fluorescent protein are respectively selected from the following: red fluorescent protein, green fluorescent protein, blue fluorescent protein, and yellow fluorescent protein.

7. The dual fluorescence detection method for detecting gene expression in filamentous fungi according to claim 1, characterized in that: When a gene for expressing a first fluorescent protein is inserted into the first insertion site of the filamentous fungus, and a gene for expressing a second fluorescent protein is inserted into the second insertion site of the filamentous fungus, homologous recombination is used for insertion.

8. A method for expressing an exogenous gene, characterized in that: Use filamentous fungi as heterologous expression hosts and insert the foreign gene to be expressed into any of the following sites: (1) Site 2: The upstream and downstream genes in Aspergillus nidulans are AN0716 and AN9436, and the chromosome position of the site in Aspergillus nidulans is ChrVIII: 2657115-2689534; (2) Site 6: The upstream and downstream genes in Aspergillus nidulans are AN4560 and AN4777, and the chromosomal location of the site in Aspergillus nidulans is ChrIII: 1022424-1662379; (3) Locus 20: The upstream and downstream genes in Aspergillus nidulans are AN0104 and AN0067, and the chromosome position of the locus in Aspergillus nidulans is ChrVIII: 4602068-4707084; (4) AU1 locus: The upstream and downstream genes in Aspergillus nidulans are AN8385 and AN8378, and the chromosome position of the locus in Aspergillus nidulans is ChrIII: 293891-319970; (5) F9 locus: The upstream and downstream genes in Aspergillus nidulans are AN7916 and AN7905, and the chromosome position of the locus in Aspergillus nidulans is ChrIII: 198368-226926; (6) MON locus: The upstream and downstream genes in Aspergillus nidulans are AN0153 and AN10039, and the chromosomal position of the locus in Aspergillus nidulans is ChrIII: 4435547-4460859.

9. The method for expressing exogenous genes according to claim 8, wherein: The filamentous fungus is Aspergillus, Penicillium or Trichoderma.

10. The method for expressing exogenous genes according to claim 9, characterized in that: The filamentous fungus is Aspergillus niger (A. niger), Aspergillus nidulans (A. nidulans), Aspergillus oryzae (A. oryzae), Aspergillus fumigatus (A. fumigatus) or Aspergillus awamori (A. awamori) of the genus Aspergillus; or the filamentous fungus is Penicillium citrinum of the genus Penicillium; Or the filamentous fungus is Trichoderma reesei of the genus Trichoderma.

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